Modularisation of novel multi-enzyme cascades and synthesis strategy development

Fischöder, Thomas; Elling, Lothar (Thesis advisor); Schwaneberg, Ulrich (Thesis advisor)

Aachen (2019)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2019

Abstract

This thesis is purposed to be a small contribution to the unique progress of understanding biological processes and a beneficial application of this certain knowledge. The enzymatic glycan synthesis is an attractive field for medicine and nutrition in this regard. The scope of this thesis focuses, on the one hand, on the development of synthesis strategies to unlock an enzymatic glycan tailoring in relevant scales and suitable amounts for further reasonable applications and, on the other hand, on a modularisation of novel multi-enzyme cascades, to facilitate a quick customization response and innovative abilities. Nucleotide sugars are essential substrates for Leloir-glycosyltransferases (GTs). The deployment of nucleotide sugar producing multi-enzyme cascades into a repetitive batch approach, in case of this thesis, substantially enhances their productivities and offers thereby the chance to overcome the substrate bottleneck for GT-based synthetic routes. A novel enhanced expressed GT fusion construct, implemented into the respective enzyme module (EM), now facilitates the production of LacNAc type I glycans in suitable amounts for reasonable further applications, here the chemo-enzymatic synthesis of novel neo-glycoproteins. Binding studies on these synthetic ligands gave the first evidence of a glycan-specific selectivity for galectin-3 and its truncated counterpart (Gal-3Δ). Modularised GT EMs were set-up in an expedient assembly line towards linear HMOS and utilized in an efficient semi-sequential one-pot synthesis approach. Thereby, a broad set of HMOS as reference standards including several previously not available structures were provided here. A further developed assembly line set-up combines nucleotide sugar producing EMs and the respective GT EMs. Operated in a sequential synthesis approach it facilitates i.a. the provision of well defined isotopically labelled HMOS. Their further utilization as quantification standards for a MALDI-TOF-MS-based absolute quantification of HMOS was demonstrated here within a proof-of-concept exercise. In regard to the claim of this thesis, a considerable set of novelties were reached as potentially valuable contributions for prospective works. Improved substrate access may pave the way for an establishment of GT-based synthesis approaches as straightforward efficient "routine" method. Novel findings and synthetic routes allow prospective application developments for tracing Gal-3Δ during tumour progression or therapies. Tailored novel reference standards support a much easier identification of by now database annotated HMOS structures and tailored isotopic quantification standards provide the chance to close the analytical gap for a "routine" absolute quantification method.

Institutions

• Department of Biology [160000]
• Laboratory for Biomaterials [162820]